Among the various transcription factors that associate with Smads, a small group, the proto-oncogenes c-ski
, exhibit broad negative regulation of most TGF-β superfamily responses (30
). Another group of Smad-interacting corepressors, TGIF and Tob, selectively inactivate the TGF-β and BMP pathways, respectively (55
). Here we report the first repressor of TGF-β- and BMP-specific Smad pathways that exhibits functional and target gene selectivity.
YY1 can repress specific gene targets of both TGF-β and BMP pathways that depend on Smad binding to SBE-like DNA, such as PAI-1
(Fig. ). Both of these genes contain multiple SBEs scattered in their promoter regions, many of which contribute to the maximal induction of the genes by the TGF-β/BMP/Smad signal (9
). Thus, multiple Smad complexes with the promoter DNA may be critical for proper gene induction, and YY1 appears to be able to interfere with this Smad-dependent transcriptional mechanism. On the other hand, YY1 does not affect the responsiveness of genes such as p15
, and c-myc
to TGF-β (Fig. ). For the Smad-mediated regulation of these three promoters, Smads need to form complexes with other transcription factors, such as Sp1, Miz-1, or p107/E2F4/5, that associate with the responsive sites on the promoter (7
). Neither the p15
, the p21
, nor the c-myc
gene contains multiple SBEs scattered in the responsive areas of its promoter. Furthermore, the p21
promoter can be induced by Smad3 and Smad4 mutants that are defective in SBE binding (40
). Consequently, YY1 cannot effectively block the Smad-dependent regulation of these genes.
Alternatively, the repressive effect of YY1 may depend on its ability to be recruited to the target promoter independent of Smad proteins. Among the promoters we have analyzed, only c-myc
has been previously reported to be regulated by YY1 (43
), and a number of observations favor a model of YY1 interference with the Smad pathway via a protein-protein-dependent mechanism. In vitro EMSA with recombinant Smad and YY1 proteins showed an inhibitory activity for YY1 against Smad-SBE binding (Fig. ). This occurred in the absence of YY1 binding to the same DNA sequences (Fig. ). Furthermore, this effect mimicked rather well the in vivo inhibition of Smad-SBE binding (Fig. ) or Smad-PAI-1
chromatin recruitment (Fig. ), suggesting that nuclear complexes between Smads and YY1 may be sufficient for repression of Smad-dependent and SBE-containing gene promoters. Finally, YY1 and Smads interact via their respective DNA-binding domains (Fig. and ), and thus it is possible that the YY1-Smad complexes are distinct from YY1-DNA complexes at other genomic sites. Since in this study, only a limited set of responsive genes and promoters was tested, a large-scale analysis of gene expression downstream of TGF-β and BMP signaling in the presence of YY1 is an important step to undertake in the future. This may reveal additional and more complex mechanisms of cross talk between these two protein factors.
YY1 interacts with the conserved N-terminal MH1 domain of Smad4, more weakly with the MH1 domains of Smad1 and Smad3, and even more weakly with Smad2 (Fig. ). This selective interaction with Smad4 distinguishes YY1 from all other known repressors of Smads, which preferably associate with the C-terminal MH2 domain or the MH1 domain of an R-Smad (4
). The MH1 domain provides specificity for DNA binding and nuclear localization to the Smads (25
). In agreement with these conclusions, Smad2, whose MH1 domain cannot associate with DNA or with importins (25
), interacts with YY1 very weakly (Fig. ). The selectivity for the MH1 domain is of functional importance, as all presently known Smad-interacting transcription factors that bind to the MH1 domain are DNA-binding factors (23
). YY1 is also a DNA-binding protein (47
), yet it can repress Smad transcriptional activity without binding to DNA itself (Fig. ). Rather, it appears that the DNA-binding domain of YY1, which is also known to be important for its transcriptional repressor activity, is responsible for the negative regulation of the Smad pathway through conformational changes that interfere with Smad binding to DNA (Fig. ). Since this domain of YY1 is multifunctional and is also regulated by alternating acetylation-deacetylation (58
), it is possible that more complex mechanisms may govern the repressive effect of YY1 on the Smad pathway. Efforts to correlate the repression mediated by YY1 on Smads via inhibition of coactivators of the CBP family or via recruitment of histone deacetylases were not successful (data not shown).
The apparent specificity of YY1 in antagonizing cellular differentiation (Fig. and ) but not proliferative responses (Fig. ) to TGF-β family ligands correlates with the selectivity that YY1 exhibits by repressing only specific gene targets of the Smad pathway (Fig. , , and ). This hypothesis is consistent with the complex transcriptional functions of YY1 that depend on the promoter and cellular context. The distinct antagonism that YY1 exhibits towards cell differentiation regulated by TGF-β or BMP is consistent with the previously described involvement of YY1 in cellular differentiation (47
). A novel action of YY1 in EMT and preosteoblastic differentiation is established here, in addition to the role of YY1 as a negative regulator of myocyte differentiation (52
). The negative effect of YY1 against TGF-β-induced EMT (Fig. ) and the neutral effect of YY1 on cell growth (Fig. ) suggest that YY1 may act as an antitumor agent with respect to the action of TGF-β in cancer progression. TGF-β is known to act as a tumor suppressor due to its growth-inhibitory effects on many cell types but also as an enhancer of tumor progression based on its ability to induce EMT, tumor cell invasiveness, immune cell suppression, and angiogenesis (10
). YY1 interferes with one of the protumorigenic actions of TGF-β, EMT, yet it leaves a tumor-suppressing action (growth inhibition) intact, which would make it a suitable therapeutic target in cancer cases with a strong contribution of the TGF-β pathway.
It is attractive to suggest that during TGF-β or BMP signaling, the activity of YY1 is regulated negatively to allow efficient activation of gene targets that lead to cell differentiation. However, we have so far been unable to obtain convincing evidence that TGF-β superfamily signaling leads to regulation of YY1 protein levels (data not shown). The antisense and siRNA experiments provide evidence that YY1 may define threshold levels of TGF-β or BMP signaling (Fig. and ). The association of YY1 with specific nuclear Smad complexes, and in particular the nuclear Smad4 pool, may offer the cell a mechanism to titrate TGF-β or BMP signaling towards specific gene targets. According to this model, Smad4 plays a crucial role in defining the strength and/or length of the nuclear signal induced by TGF-β family members.
In summary, the identification of YY1 as a Smad-interacting protein that negatively regulates TGF-β superfamily signal transduction leading to cellular differentiation opens novel ground for future analysis of the complex mechanisms that integrate diverse extracellular signals to choices of cellular fate.